Biology of Prion Protein and the TSE Diseases

Pron病或传染性海绵状脑病是人和动物的传染性神经退行性疾病。Prion病的一个主要特征是将正常的宿主蛋白Prion蛋白(PrP)折叠和聚集成疾病相关的蛋白酶抵抗形式(PrPres)，这可能会导致脑损伤。 在2012财年，我们研究了在无细胞PMCA系统中产生抗蛋白酶的PrPres蛋白。在PMCA的每个周期，我们量化了产生的PrPre感染量，并将其与PrPres产生的量进行了比较。4个周期的传染性产出是投入的320倍，6个周期和8个周期的传染性产出分别是投入的3200~1万倍。这些数据表明，在没有ATP和二价阳离子的情况下，无细胞系统中产生了越来越多的传染性，这有力地支持了普恩病毒感染性的媒介不是病毒的假设。然而，PMCA与脑源性样本相比，传染性滴度与PrPres数量的比率较低，这表明体外产生的相当数量的PrPres可能是非传染性的。 在2012财年的其他实验中，我们研究了培养的星形胶质细胞和小胶质细胞在瘙痒脑提取物刺激后对细胞因子释放的刺激作用。我们之前曾报道，感染普恩病毒的小鼠脑匀浆在原代培养的星形胶质细胞和小胶质细胞中诱导细胞因子蛋白释放。在这里，我们测量了培养的胶质细胞释放的细胞因子，以确定感染脑中的哪些因素有助于小胶质细胞和星形胶质细胞的激活。在分析细胞因子释放的分析中，无论是从感染的小鼠脑中提纯的PrPres还是在体外产生的PrP淀粉样纤维都不能在体外刺激胶质细胞。然而，在缺乏PrPreS的澄清的瘙痒病脑匀浆中存在的一种不稳定的蛋白酶敏感蛋白诱导了显著的神经胶质刺激。在瘙痒病脑刺激星形胶质细胞后，检测到八种不同的细胞因子，但在使用小胶质细胞的类似实验中，只检测到两种。 在2012财年，我们还研究了一个转基因小鼠系统，其中将自然发生的人PrP(N171S)变异插入到小鼠的PrP中。表达该突变的小鼠对ME7和22L羊瘙痒病敏感，但90%的小鼠对RML和79A耐药。剩下的10%的突变小鼠发病很晚，大脑PrPres水平很低。令人惊讶的是，在使用PMCA的无细胞体外实验中没有看到这种耐药性。这个转基因小鼠系统可能是构象选择模型的一个例子，在该模型中，某些PrP菌株的结构不利于与表达某些多态的PrP分子相互作用。

Prion diseases or transmissible spongiform encephalopathies are infectious neurodegenerative diseases of humans and animals. A major feature of prion diseases is the refolding and aggregation of a normal host protein, prion protein (PrP), into a disease-associated protease-resistant form (PrPres) which may contribute to brain damage. In FY12 we studied the generation of protease-resistant prion protein (PrPres) in a cell-free PMCA system. At each cycle of PMCA we quantitated the amount of prion infectivity produced and compared this to the amount of PrPres generated. The output of infectivity after 4 cycles was found to be 320-fold greater than the input, and was 3200 to 10,000 fold greater than input after 6 and 8 cycles respectively. These data showing generation of increasing amounts of infectivity in a cell-free system in the absence of ATP and divalent cations provided strong support for the hypothesis that the agent of prion infectivity is not a virus. However, the ratio of the infectivity titer to the amount of PrPres was lower in PMCA versus brain derived samples suggesting that a substantial amount of PrPres generated in vitro might be non-infectious. In other experiments in FY12 we studied the stimulation of cytokine release by cultured astroglia and microglia after stimulation by scrapie brain extracts. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing cytokine release, glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or PrP amyloid fibrils produced in vitro. However, significant glial stimulation was induced by a heat labile protease-sensitive protein present in clarified scrapie brain homogenates lacking PrPres. Eight different cytokines were detected after scrapie brain stimulation of astroglia but only two were detected in similar experiments using microglia. In FY12 we also studied a transgenic mouse system where a naturally occurring variation of human PrP (N171S) was inserted into PrP of mice. Mice expressing this mutation were susceptible to mouse scrapie strains ME7 and 22L, but ninety percent of the mice were resistant to strains RML and 79A. The remaining 10% of mutant mice developed disease very late and had low levels of brain PrPres. Surprisingly this resistance was not seen in cell-free in vitro experiments using PMCA. This transgenic mouse system may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.